Substrate-specific protein degradation is central to many cellular regulatory mechanisms. Rapidly degraded regulatory proteins include many transcription factors, proto-oncogene products, tumor suppressors, and cell cycle regulators. However, the molecular features that distinguish such naturally short-lived proteins from stable ones remain largely unknown, and it is only recently that the pathways responsible for their degradation have begun to be deciphered. Eukaryotes have a complex enzymatic system for the covalent ligation of proteins to ubiquitin (Ub) and for their subsequent degradation by a large protease called the proteasome. The "Ub-proteasome system" is now known to be responsible for the bulk of regulated proteolysis in eukaryotic cells. Moreover, the etiology and pathology of many diseases, e.g., neurodegenerative disorders such as Alzheimer's and Parkinson's disease, diabetes, and both sporadic and hereditable forms of cancer are associated with abnormalities in the Ub-proteasome system; these connections are just beginning to be investigated at a mechanistic level. Thus, one can expect that a deeper understanding of Ub-proteasome-dependent proteolysis will have a substantial impact on our ability to diagnose and treat a large number of serious human medical problems. Many basic features of Ub-protein ligation and of proteasome action and assembly are not understood. In this renewal, studies are described that address the functions and mechanisms of Ub-dependent proteolysis in genetically manipulatable eukaryotes, the yeast S. cerevisiae. The proposal is divided into two parts. The first is directed toward understanding the degradation by the Ub-proteasome pathway of a naturally short-lived model protein, Mata2 (Aims1-2). In the second section, experiments that dissect proteasome biogenesis and function are proposed (Aims 3-4): Aim 1. Characterize the recently discovered Doa 10 protein and its role in targeting proteins for degradation. Aim 2. Investigate the Ubc4/Ubc5-dependent ubiquitination of Mata2 and examine physiological aspects of Mata2 degradation. Aim 3. Use biochemical and genetic approaches to dissect the function of the Doa3/beta5 proteasome subunit pro-peptide, which is essential for proteasome assembly in vivo. Aim 4. Continue structure-function studies on the yeast 20S proteasome and assess its interactions with the l9S regulatory complex.